Identification of serine hydrolases involved in Plasmodium host cell invasion

鉴定参与疟原虫宿主细胞侵袭的丝氨酸水解酶

• 批准号: 8594762
• 负责人: Brooke Anderson-White
• 金额: $ 4.92万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8594762
• 关键词: Active Sites; Affect; Affinity Chromatography; Automobile Driving; Binding; Biological Assay; Blood; Carbamates; Case Study; Cell physiology; Cells; Cessation of life; Data; Defect; Development; Disease; Drug Targeting; Drug resistance; Enzymes; Erythrocytes; Family; Generic Drugs; Growth; Hepatocyte; Human; Hydrolase; Hydrolysis; In Vitro; Infection; Knock-out; Label; Lead; Libraries; Ligands; Liver; Malaria; Mammals; Mass Spectrum Analysis; Metabolism; Methods; Modification; Mus; Mutate; Organism; Parasites; Peptide Hydrolases; Plasmodium; Plasmodium berghei; Plasmodium falciparum; Play; Prevalence; Proteins; Proteolysis; Proteome; Recombinant Proteins; Relapse; Reporting; Research; Role; Serine; Serine Hydrolase; Serine Protease; Site; Solid; Staging; Structure; Structure-Activity Relationship; Subtilisins; System; Therapeutic; Triazoles; Urea; activity-based protein profiling; base; disorder control; drug development; experience; in vivo; inhibitor/antagonist; interest; novel; novel therapeutics; obligate intracellular parasite; profilin; public health relevance; screening; small molecule; success; therapeutic target

DESCRIPTION (provided by applicant): Plasmodium falciparum is the parasite responsible for malaria, a disease that continues to devastate all tropical regions of the world. A quarter of a million cases of malaria are reported annually with close to a million of these cases resulting in death. Efforts to control this disease are complicated by growing drug resistance in the parasite, leading to an imminent need for new drug targets. One avenue to the identification of novel targets is to discover and characterize essential enzymes in the parasite by inhibiting them with small molecules. The serine hydrolases are a large family of enzymes with highly reactive and covalently modifiable active sites making them attractive for inhibition with small molecules. Furthermore serine hydrolases are well represented in most organisms including humans where they comprise 1% of all proteins. In P. falciparum two essential serine hydrolases have already been identified, subtilisin-like serine protease 1 (PfSUB1) and 2 (PfSUB2). In order to find additional essential serine hydrolases in the parasite, a library of highly effective serine hydrolase inhibitors in mammals was screened in P. falciparum. This library of triazole urea compounds revealed several compounds that slow or block the growth of P. falciparum. The most efficacious compound, AA691, appears to induce a host cell invasion defect. Furthermore it has been verified that AA691 binds to at least one of the same targets as fluorophosphonate (FP), a generic serine hydrolase inhibitor, in competition assays suggesting this defect is due to inhibition of serine hydrolases. In this project the serine hydrolases inhibited by AA691 will be isolated using a probe based on the structure of AA691 that covalently modifies its targets. The targets will then be identified using tandem orthogonal proteolysis-activity-based protein profilin (TOP-ABPP) and mass spectrometry. The role(s) of these targets in host cell invasion will be characterized in culture through the creation of direct and regulatable conditional knockouts in P. falciparum. The invasion defect will be further verified in vivo with P. berghei mouse infections. Finally the effects of AA691 on the liver stage will be assessed in hepatocyte culture and in vivo. The rising prevalence of P. vivax infection, which remains dormant in the liver leading to relapse after blood stage treatment, is driving a push for therapeutics against liver stage parasites. There is an urgent need for novel malaria therapeutics and the results of this research could lead directly to the development of these drugs.

描述（由申请人提供）：恶性疟原虫是导致疟疾的寄生虫，疟疾是一种继续在世界所有热带地区肆虐的疾病。每年报告的疟疾病例有25万例，其中近100万例导致死亡。控制这种疾病的努力由于寄生虫中日益增长的耐药性而变得复杂，导致迫切需要新的药物靶点。鉴定新靶点的一个途径是通过用小分子抑制寄生虫中的必需酶来发现和表征它们。丝氨酸水解酶是一个大家族的酶，具有高度反应性和共价修饰的活性位点，使它们对小分子抑制有吸引力。此外，丝氨酸水解酶在包括人类在内的大多数生物体中有很好的代表性，其中它们占所有蛋白质的1%。在恶性疟原虫中，已经鉴定出两种必需的丝氨酸水解酶，枯草杆菌蛋白酶样丝氨酸蛋白酶1（PfSUB 1）和2（PfSUB 2）。为了在寄生虫中发现另外的必需丝氨酸水解酶，在恶性疟原虫中筛选哺乳动物中高效丝氨酸水解酶抑制剂的文库。该三唑脲化合物库揭示了几种减缓或阻断恶性疟原虫生长的化合物。最有效的化合物AA 691似乎诱导宿主细胞侵袭缺陷。此外，在竞争试验中已经证实，AA 691与氟膦酸酯（FP）（一种通用丝氨酸水解酶抑制剂）结合至少一种相同的靶标，表明该缺陷是由于丝氨酸水解酶的抑制所致。在本项目中，将使用基于AA 691结构的探针（共价修饰其靶标）分离AA 691抑制的丝氨酸水解酶。然后将使用串联正交蛋白水解活性为基础的蛋白质profilin（TOP-ABPP）和质谱法鉴定靶点。这些靶标在宿主细胞侵袭中的作用将在培养物中通过在恶性疟原虫中产生直接和可调控的条件性敲除来表征。侵袭缺陷将在伯氏疟原虫小鼠感染的体内进一步验证。最后，将在肝细胞培养物和体内评估AA 691对肝脏阶段的影响。间日疟原虫感染的患病率不断上升，在肝脏中保持休眠状态，导致血液阶段治疗后复发，这推动了针对肝脏阶段寄生虫的治疗方法。迫切需要新型疟疾治疗方法，这项研究的结果可能直接导致这些药物的开发。